98% of the mammalian genome is non-coding, a significant fraction of which has cis- regulatory function. If we are ever to interpret the effects of the vast number of non-coding polymorphisms in the human population, we must gain a more complete understanding of the cis-regulatory grammar of gene expression. Fueled by genomic-scale technologies, the identification of potential cis-regulatory elements (CREs) is proceeding rapidly. Unfortunately, the rate of discovery of putative CREs far outpaces our capacity to functionally analyze them in vivo. Clearly, there is an urgent need for new high-throughput technologies to elucidate the functional architecture of CREs in mammalian cells. Reporter gene analysis is the standard assay for dissecting the function of mammalian CREs, but the time- and labor-intensive nature of the assay makes it impractical for testing large numbers of CREs. We propose to harness the power of Next Generation Sequencing (NGS) to create a system for the multiplexed analysis of reporter genes in mammalian cells. Our strategy is to fuse libraries of CREs to barcoded reporter genes and quantify their output by NGS. We will demonstrate the utility of this assay by studying cis-regulation in a mammalian neuronal cell type, the retinal photoreceptor cell. In Aim 1 we will dissect, at single nucleotide resolution, the photoreceptor- specific Rhodopsin (Rho) promoter. More than 15,000 mutant promoters will be assayed in a single, multiplexed assay in living retinas, an experiment that is not possible with any existing methodologies. The data from this experiment will allow us, for the first time, to quantify the relationship between evolutionary sequence conservation and cis-regulatory activity at nucleotide resolution. In Aim 2 we will deploy the assay to test 1,000 Chip-seq peaks that are enriched for binding to the key photoreceptor transcription factor, Crx. In a single, multiplexed experiment we will test all 1,000 genomic sequences for their ability to drive photoreceptor- specific expression in vivo. In addition, in Aim 2 we will assay combinatorial libraries of synthetic promoters composed of binding sites for Crx and two other transcription factors, Nrl and Nr2e3, known to play a key role in controlling photoreceptor-specific gene expression. The output of all these experiments will be analyzed using a formal thermodynamic model of combinatorial cis-regulation. Our intention is to demonstrate, in a relatively short time frame, that it is possible to unravel the cis-regulatory grammar of an important mammalian cell type in vivo. This study will serve as a proof-of-concept that CRE analysis can be made as high- throughput as CRE discovery.